Abstract

Animal models are indispensable for the study of glomerulonephritis, a group of diseases that destroy kidneys but for which specific therapies do not yet exist. Novel interventions are urgently needed, but their rational design requires suitable in vivo platforms to identify and test new candidates. Animal models can recreate the complex immunologic microenvironments that foster human autoimmunity and nephritis and provide access to tissue compartments not readily examined in patients. Study of rat Heymann nephritis identified fundamental disease mechanisms that ultimately revolutionized our understanding of human membranous nephropathy. Significant species differences in expression of a major target antigen, however, and lack of spontaneous autoimmunity in animals remain roadblocks to full exploitation of preclinical models in this disease. For several glomerulonephritides, humanized models have been developed to circumvent cross-species barriers and to study the effects of human genetic risk variants. Herein we review humanized mouse prototypes that provide fresh insight into mediators of IgA nephropathy and origins of antiglomerular basement membrane nephritis and Goodpasture's disease, as well as a means to test novel therapies for ANCA vasculitis. Additional and refined model systems are needed to mirror the full spectrum of human disease in a genetically diverse population, to facilitate development of patient-specific interventions, to determine the origin of nephritogenic autoimmunity, and to define the role of environmental exposures in disease initiation and relapse.

glomerulonephritis

translational

humanized model

glomerulonephritis (gn) is the third most common cause of end stage renal disease (ESRD) in the U.S. and underlies chronic kidney disease in over 40% of patients in some countries (28). In ESRD patients who receive kidney transplants, allograft loss is attributable to recurrent or de novo GN in a significant proportion of patients (6). Yet, for most patients who develop GN the precipitating cause is unknown, the underlying mechanism is poorly understood, and available therapies are nonspecific, toxic, and of variable efficacy. Novel targeted interventions are urgently needed, but their rational design requires suitable platforms to dissect disease pathogenesis, identify critical pathways, and test candidate treatments.

Animal models have proven invaluable and are currently irreplaceable for this purpose. Important tissues are frequently inaccessible in patients or their acquisition carries substantial risk of injury. In vivo experimentation is often not safe in humans, whereas judicious, ethical manipulation using animal models permits important insights not otherwise obtainable. In vivo modeling is particularly relevant for GN because of its immunological basis. Immunity develops over time from dynamic interactions of multiple cell types and soluble mediators within diverse and complex microenvironments–a situation that cannot be reproducibly replicated in vitro. This is particularly true of autoimmunity, the onset and course of which are heavily influenced by gene-environment interactions and which underlies most GN. Circulating or deposited autoantibodies (autoIg) are key to diagnosis and are suspected mediators in multiple GN, including anti-glomerular basement membrane (GBM) GN, IgA nephropathy (IgAN), membranous nephropathy (MN), lupus nephritis, and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), in which approximately three-quarters of patients develop GN.

The contribution of animal models to our current understanding of human GN is indisputable and perhaps most aptly illustrated by primary MN. Careful study of rat Heymann nephritis, which pathologically and clinically closely mirrors human MN, revealed a critical role for anti-podocyte antibodies in pathogenesis. This gave impetus to the ultimately successful decades-long search for analogous in situ mechanisms and human podocyte antigens (Ag) in humans (3, 15, 17, 67). M-type phospholipase A2 receptor 1 (PLA2R1) was identified as the target of circulating and deposited IgG in 70% of patients with primary MN (3). However, there is as yet no definitive proof that anti-PLA2R1 autoIg are pathogenic. Passive transfer of patient autoIg to rodents did not induce MN, consistent with the absence of PLA2R1 expression in rodent glomeruli (16). Attempts to develop a mouse model of this common form of MN by targeted expression of human PLA2R1 in mouse podocytes have been unexpectedly challenging (5). When it becomes available, such a tool will help resolve questions regarding anti-PLA2R1 pathogenicity and provide insight into the disease relevance of PLA2R1 risk variants (61) as well as provide a platform for preclinical efficacy studies based on PLA2R immunodominant epitope-based therapies (20, 30). Moreover, a dual humanized model based on coexpression of human PLA2R1 with a humanized immune system will provide a means to address multiple additional questions regarding MN pathogenesis in humans: the role of HLA-DQA1 high-risk alleles (61); the origin of the IgG4 predominance observed among patients' anti-PLA2R IgG, noting that IgG4 is not found in wild-type mice; the basis of mutual exclusivity in serological responses to PLA2R and thrombospondin type-1 domain-containing 7A (THSD7A), the podocyte Ag target in ∼10% of MN patients (67); and whether full blown human MN requires collaboration of additional anti-podocyte autoIg, similar to the anti-integrin receptor and anti-glycoplipid Ig that contribute to proteinuria and complement activation in Heymmann nephritis (63). Notably, mouse podocytes do express THSD7A (21), and injection of MN patients' anti-THSD7A autoIg into naïve BALB/c mice leads to human IgG colocalization with nephrin and induction of proteinuria and MN-like histopathologic changes (68). This supports a causative role for circulating anti-podocyte autoIg in human MN pathogenesis and provides a new model to dissect disease mechanisms.

It is clear, however, that no one animal model will recapitulate the full spectrum of human MN. It is well-established that human MN can arise from diverse immune responses and mechanisms, subject to genetic modification (3, 15, 17, 61, 67). MN glomerular immune deposits form by mechanisms in addition to direct binding by anti-podocyte autoIg. In childhood MN, pathogenesis is persuasively attributed to a nonautoimmune in situ mechanism that involves deposition of circulating anti-bovine-albumin IgG that bind dietary cationic bovine serum albumin (presumably absorbed from ingested cow's milk) that plants on anionic sites of the glomerular filter (17). Thus development of patient-specific interventions will require study of multiple distinct animal models. In this regard, mouse MN models that rely on passive transfer of heterologous anti-mouse podocyte Ig or that are based on immunization with heterologous cationic albumin or α3(IV)NC1 collagen are likely to provide novel and fundamental insights into Ag targets, disease mechanisms, and genetic modulation that will be applicable to different subsets of MN patients (8, 42, 76).

Below we review recent progress in modeling three additional GN, IgAN, anti-GBM GN, and ANCA vasculitis, characterized by a prominent humoral autoimmune component. The focus is on recent modeling in mouse, as a tractable species particularly amenable to genetic engineering and humanization for in vivo mechanistic studies, with an emphasis on new insights, limitations, and opportunities to increase translational value. For updates on the role of animal models in understanding C3 nephropathies and lupus nephritis, additional diseases for which animal models have been critical in understanding pathogenesis in humans, the reader is referred to recent excellent reviews (1, 52).

IgA Nephropathy

IgA Nephropathy (IgAN) is the most common primary GN in the world and a major cause of chronic kidney disease and ESRD, yet it has no specific curative therapy and an incompletely understood pathogenesis. Patients' circulating and deposited IgA are typically polymeric IgA1 bearing galactose-deficient O-glycans targeted by anti-glycan IgG autoIg. These observations, clinical features (recurrence in allografts and synpharyngitic hematuria), and capacity of bone marrow or splenocytes to transfer IgAN-like disease in the experimental setting suggest a systemic and mucosal lymphoid origin, although clinical studies have yet to identify a convincing pathogenetic link to mucosal disease. Multiple rodent models have been developed based on dietary, oral, or respiratory tract Ag exposure or genetic manipulation of candidate genes. Most of these models demonstrate elevated IgA levels and mesangial IgA deposits but no or limited renal injury (64). These models may nonetheless have clinical relevance to the spectrum of disease in human IgAN, because isolated hematuria or proteinuria are primary manifestations in a prominent subset of patients.

A recently developed mouse model of spontaneous IgAN, termed early onset grouped ddY, demonstrates more reproducible and robust renal histopathologic injury, similar to that in more severely afflicted IgAN patients (47). Generated by interbreeding early onset ddY outbred mice, grouped ddY mice develop mesangial IgA, IgG, and C3 deposits and proliferation, proteinuria, and renal failure. Four genetic loci associate with disease in this model, including regions syntenic to susceptibility genes in human IgAN (65). Thus this model should provide insight into IgAN immunopathologic mechanisms and genetic susceptibility relevant to humans. However, novel therapies aimed at specific glycation enzymes or glycan structures will likely require a more human-like model.

Species differences in structure, function, and expression of IgA and myeloid IgA receptor, FcαR (CD89), have raised concerns about the clinical relevance of mouse spontaneous IgAN. Only humans and hominoid primates have the IgA1 subclass; rodents have a single IgA gene and lack the IgA1-like structure. Many mouse IgA also lack hinge region O-glycans, although strains carrying the Igh-2a and Igh-2c allotypes have a putative hinge O-glycosylation site (49, 55). Serum IgA circulates almost exclusively in its monomeric form in healthy humans, whereas serum IgA is predominantly polymeric in mouse (12). Human IgA activates complement by the alternative and lectin pathways (57), whereas mouse IgA may lack this capacity (2). Mice also lack a homologue of the myeloid FcαR CD89 that has been implicated in pathogenic immune complex formation and function in IgAN (32).

The dual humanized Hu-IgA1-KI/CD89Tg mouse model of Monteiro and colleagues (4) represents a significant breakthrough in developing an IgAN model more faithful to the human phenotype. Targeted knock-in of the human Ig α1 gene at the host IgM switch locus replaces most serum Ig with monomeric human IgA1 (18), and the human CD11b promoter drives CD89 expression on mouse monocytes and macrophages (32). The dual humanized model capitalizes on species differences in IgA affinity for human CD89, as well as the ability of human IgA1 to engage the murine transferrin receptor 1 (TfR1, also known as CD71), a polymeric IgA receptor (12). IgA1-KI/CD89Tg mice develop spontaneous IgAN characterized by circulating IgA1/sCD89 immune complexes, hematuria, proteinuria, mesangial IgA1, C3, and CD89 deposits, renal macrophage infiltration, and upregulated mesangial cell TfR1 and transglutaminase 2 (TG2) (4). An elegant series of experiments point to soluble CD89, TfR1, and TG2 cross regulation within an autoamplifying inflammatory loop and formation of mesangial cell surface IgA1/sCD89/TfR1/TG2 multimolecular complexes in disease pathogenesis in this model. Importantly, examination of patients' serum and kidney biopsies suggests that similar mediators operate in human IgAN. Several features of severe human IgAN are absent from the model, including glomerular codeposits of IgG and altered renal function. Additionally, the single mutant α1-KI mouse develops a distinct glomerular injury with diffuse endocapillary GN and glomerular deposits of human IgA1 that colocalize with mouse vascular CD31 (4), suggesting the potential for a confounding source of renal injury. Nonetheless, the model holds promise for revealing novel pathogenic pathways relevant to human IgAN and for testing interventions, as recently described for modulation of dietary gluten (50).

Autoimmune Anti-GBM Nephritis and Goodpasture's Disease

Autoimmune anti-GBM GN and its systemic counterpart Goodpasture's (GP) disease are rare clinical entities that have been the focus of intense investigation for several reasons. Clinical manifestations can be severe, including rapidly progressive and irreversible renal failure and catastrophic pulmonary hemorrhage, and unlike most GN, the dominant target Ag, the noncollagenous 1 (NC1) domain of α3(IV)NC1 collagen, has been well characterized (51). Diagnosis depends on identification of anti-GBM autoIg either in the circulation or deposited in a linear basement membrane pattern in the kidney or lung. A pathogenic role for the anti-GBM autoIg was confirmed by classic experiments in which GN was induced in squirrel monkeys after transfer of IgG eluted from patients' kidneys (34). These early experiments also focused attention on humoral autoimmunity as a critical mediator of human GN and provided original support for therapy directed at autoantibody removal using plasmapheresis. Thus anti-GBM GN is considered a prototypic kidney-specific autoimmune disease amenable to pathogenic dissection, with the expectation that principles and mechanisms revealed will be applicable or translatable to other GN and autoimmune diseases.

Yet the major rodent models used to study autoimmune anti-GBM GN have limitations. Experimental autoimmune GN, or EAG, is induced by active immunization, using GBM or isolated or recombinant α3(IV)NC1 collagen. Investigators report variable success in inducing EAG and less commonly pulmonary hemorrhage in mice. This is partly genetic, as strain differences in disease susceptibility are reported and likely reflect both variable capacity to generate and activate pathogenic lymphocytes of appropriate specificity and IgG subclass and differences in end-organ susceptibility to inflammation and injury (25, 29). Genetic deficiency of Fcγ receptor IIB (FcγRIIB), an inhibitory Fcγ receptor expressed on B cells, macrophages, and other Ag-presenting cells (APC), permits induction of severe anti-GBM GN and acute lung injury in some settings (46).

Recent experiments suggest that Ag accessibility is also a critical disease determinant in anti-GBM GN and GP disease. Luo et al. (38) demonstrated that infusion of pathogenic anti-GBM IgG from patients into mice does not produce linear GBM Ig deposits. Cross-reactive pathogenic epitopes are clearly present in mouse GBM, but these are only revealed after in vitro acid-urea treatment of kidney sections and immunoblot of dissociated collagen IV hexamers (38). This in vivo difference with lack of binding of Goodpasture patients' IgG to murine GBM compared with avid binding reported in squirrel monkeys, in retrospect a serendipitous choice of host species in 1967 (34), is attributed to species differences in extent of NC1 hexamer cross linking. Pathogenic GP epitopes are partially buried within the collagen IV NC1 hexamer, which is stabilized by up to six peroxidasin-catalyzed covalent sulfilimine bonds that cross-link NC1 domains at the NC1 trimer interface (69). GP epitopes within highly cross-linked hexamers, including those in mouse, are not accessible even to high-affinity GP anti-GBM IgG, whereas GP autoIg can access, and even facilitate full exposure, of epitopes in noncross-linked hexamers.

In light of these findings, it is unclear what kidney and GBM Ag and epitopes are targeted by pathogenic IgG in EAG models, and in particular if the Ag are the same or different from those bound by anti-GBM patients' pathogenic IgG. The ability of immunization-induced autoIg to produce linear GBM deposits in rodents has been reinterpreted by some to reflect binding to surface-exposed “alloreactive-type” α3(IV)NC1 or hexamer epitopes. It seems likely that in some EAG rodents at least a subset of induced pathogenic autoIg ultimately can engage and deposit on GP epitopes, because epitopes may be periodically accessible during basement membrane remodeling. However, reproducible induction of EAG anti-GBM GN in mice may require modifications of the model, such as humanization of the GBM or targeted genetic, chemical, or biological disruption of enzymes or pathways that produce murine collagen IV NC1 cross links, to create a more “human-like” GBM with accessible GP epitopes. In this regard, transgenic expression of human α3(IV) collagen rescued α3(IV) collagen-deficient mice from an Alport-like nephritis phenotype (22); the status of sulfilimine cross links is not yet described. Anti-peroxidasin autoIg capable of inhibiting peroxidasin activity were recently described in a subset of GP patients (40), raising the possibility that a separate subset of autoIg may indirectly facilitate Ag exposure in some patients. A peroxidasin knockout mouse is not available and is likely embryonic lethal based on the role of peroxidasin in also stabilizing more ubiquitous collagen IV protomers. Thiocyanate is a potent in vitro inhibitor of peroxidasin-mediated cross-link formation, a notable observation in that circulating thiocyanate levels are increased by cigarette smoking, an environmental hazard implicated in GP disease (41). Timing of epitope exposure may be a critical determinant in new model development, as altered Ag access to autoIg may also reflect altered access to tolerance-susceptible lymphocytes.

These considerations also reveal a need for alternative and creative approaches or tools to assess pathogenicity of anti-GBM autoIg. Linear GBM Ig deposition is the hallmark of anti-GBM GN and GP disease in patients, yet even highly pathogenic GP anti-GBM IgG do not form linear GBM deposits in vivo in mice with stringent hexamer cross links. Thus lack of capacity to induce linear deposits (and subsequent disease) in mice cannot rule out the pathogenic potential of anti-GBM Ig. Perhaps the current most reliable, if not fully satisfactory, indicator of anti-GBM Ig pathogenicity is disease-relevant specificity, as measured using competitive inhibition of epitope binding by patients' IgG. Confirmation of in vivo nephritogenicity of true GP-like autoIg may require new models.

Animal models have also informed our understanding of the roles of cellular immunity and the major histocompatibility complex (MHC) in anti-GBM GN. T-cell reactive with α3(IV)NC1 collagen peptides presented with class II MHC molecules have been identified in patients with anti-GBM GN, healthy individuals, and immunized rodents. Current paradigms of adaptive immunity predict that pathogenic anti-GBM autoIg are produced by anti-GBM B cells activated by cognate anti-GBM CD4+ T cells. Immunodominant Ag peptides that drive the Ag-specific CD4 T-cell responses are potential therapeutic targets. However, no clear consensus α3(IV)NC1 collagen peptide emerged from study of conventional rodent models and human cells, possibly due to species differences in the MHC and peptide-processing machinery.

Ooi et al. (48) adopted a different approach by immunizing HLA-DR2+ transgenic (Tg) C57BL6 (B6) mice. The HLA DR2 heterodimer is formed by coexpression of monomorphic DRA1*0101 with the class II DRB1*1501 allele, which is carried by a striking 75–90% of anti-GBM patients (54). MHC class II proteins are expressed on the surface of B cells and other APC, as well as on thymic medullary cells that direct selection of T-cell receptors during CD4+ T-cell development. An overlapping CD4+ T-cell epitope was identified for immunized B6-HLA-DR2+ Tg mice (48) and blood leukocytes from patients with anti-GBM nephritis (7). Understanding the mechanism by which DR2 and peptides control anti-GBM GN and GP disease is crucial. An existing therapeutic agent, copolymer 1 (Cop1, glatiramer), binds DR2, inhibits APC/DR function, and is efficacious in DR2+ relapsing-remitting multiple sclerosis, an autoimmune neurological disease that is also strongly linked to DR2. However, enthusiasm for Cop1 use in patients with anti-GBM GN is tempered by reports of anti-GBM GN developing in multiple sclerosis patients treated with a different immunomodulator, anti-CD52 monoclonal antibody (mAb) (10). A dual humanized model in which HLA-DR2+Tg mice are reconstituted with human lymphocytes may be helpful in resolving this. HLA-DR2+Tg mice may also prove useful in exploring the role of lesser appreciated but potent nonclassical, peptide-independent functions of the MHC (13).

An independent role for anti-α3(IV)NC1 collagen T cells in mediation of anti-GBM GN needs further exploration. Transfer of peptide-reactive IFNγ+ CD4 effector T-cell clones derived from immunized B6-HLA-DR2+Tg mice to naïve DR2+ recipient mice produced an apparent pauci-immune kidney pathology (48). Whereas the applicability to conventional human anti-GBM GN is unclear, in that identification of either circulating or tissue-deposited anti-GBM Ig is mandatory for diagnosis of the human disease, this observation and model may provide insight into pathogenesis in the substantial subset of patients with anti-GBM/ANCA+ overlap disease.

Humanized models will be useful for probing genetic origins of the human anti-GBM response. Sequence and structural analysis of pathogenic Ig will provide insight into the structure of cognate Ag (or tolerogen) epitopes, as well as the nature of B cell populations from which the Ig emerge. Immunoglobulins function not only as soluble effectors of humoral immunity but also as Ag-specific cell surface receptors that transmit signals for cell activation and tolerance induction. Direct sequencing of patients' circulating Ag-specific Ig or B cells, however, has been unsuccessful. Epstein Barr virus transformation of patients' blood cells has failed to isolate a human mAb reactive with GP epitopes; a suitable probe to capture Ag-specific cells is not available, and accurately sequencing polyclonal IgG mixtures remains technically prohibitive. Murine anti-GBM mAb have been sequenced, but substantial species differences in Ig gene loci and B-cell repertoires advise caution in extrapolating to origins of anti-GBM Ig in humans (14). As an alternative approach, humanized mice from two models were immunized to generate human anti-α3(IV)NC1 collagen mAb (19, 43, 71). In Xenomice, mouse Ig genes are replaced with their human counterparts, whereas in Hu-HSC mice a human immune system is reconstituted by infusion of human hematopoietic stem cells (HSC) into conditioned NOD-scid-γ (NSG) recipients. Sequence analysis of the human anti-GBM mAb revealed unusual Ag binding site motifs as well as expression of the same human Ig heavy chain variable region gene by a subset of mAb derived from each model, suggesting biased selection in the human anti-α3(IV)NC1 response and potential targets for intervention.

A limitation of Hu-HSC mice as a disease model is their low level production of human IgG. This is related to lack of expression of human MHC genes in the mouse thymus, such that developing human T cells select on the mouse, not human, MHC molecules. This prevents optimal interaction of mature T cells with HLA-expressing B cells in the periphery of Hu-HSC mice. A novel dual humanized HLA-DR2+Tg/Hu-HSC model in development should eliminate this problem and permit tracking of human tolerance-susceptible IgM+ anti-GBM precursors and isotype-switched IgG effectors.

The utility of Hu-PBL mice, generated by infusion of patients' peripheral blood leukocytes (PBL) into immunodeficient recipients, for human anti-GBM mAb recovery is less certain. PBL contain mature lymphocytes that undergo potent xenoactivation in Hu-PBL mice. However, despite marked human B cell activation and differentiation, levels of circulating human anti-GBM Ig in Hu-PBL mice established using PBL from patients with active anti-GBM GN were no different than controls (71). This suggests that few if any anti-GBM B cells are circulating in patients with active disease. This is consistent with reports that Ag-specific B cells are rare among PBL, due to their brief circulation half-life and rapid migration from spleen and lymph nodes to specialized niches in bone marrow, the site from which differentiated plasma cells provide the majority of serum Ig (33, 56, 62). This dissociation of serum IgG from contemporaneous circulating lymphocytes has implications for the study of pathogenic immune cell subsets, which may reside in tissues more readily accessed in Hu-HSC mice than in patients.

The commonly used humanized models are particularly suited for investigating the proximal (inductive) limb of human pathogenic autoimmunity. They can address basic questions regarding human anti-GBM disease onset, the answers to which could guide the design of new preventative and therapeutic interventions. The site (bone marrow, spleen, kidney, lung) at which pathogenic anti-GBM autoreactivity originates is unknown, as is the nature of the precipitating event, the cause of the transient loss of tolerance that permits anti-GBM lymphocyte survival and activation, the roles of tolerogen or Ag cross reactivity and epitope spreading, and the mechanism by which environmental exposure contributes to disease.

In contrast, humanized models will require additional modifications for optimal study of the distal, or effector, arm of disease, particularly for IgG-mediated anti-GBM disease. Ig deposition on the GBM may be absent even in the face of a vigorous anti-GBM IgG response, due to masking of target epitopes behind stringent cross links, as discussed earlier. The commonly used NSG strain lacks complement factor 5, carries a mutation in the IL2 receptor γ-chain that normally supports signaling in multiple inflammatory cytokine pathways, and expresses only mouse IgG Fcγ receptor, such that human IgG engagement of inflammatory effector systems is blunted on this background. Nonetheless, ongoing modifications including serial humanization will provide progressively sophisticated platforms for mechanistic dissection and for in vivo efficacy testing. In this regard, Hu-HSC mice produced in (BALB/cx129)Rag2−/−IL2Rγ−/− recipients in which murine IL-3 and GM-CSF are replaced by their human counterparts facilitates human immune responses in lung (70), another organ targeted by pathogenic anti-GBM IgG. This or next generation models may be useful to assess immunologic consequences of cigarette smoke or inhaled aromatic hydrocarbons, two environmental exposures implicated in human GP disease.

In contrast to EAG, nephrotoxic serum nephritis (NSN) is a nonautoimmune planted foreign Ag model in which administration of anti-GBM nephrotoxic serum to susceptible animals results in a reproducible crescentic GN. A GBM-immunized donor (typically goat or rabbit) is used as the source of heterologous anti-GBM serum or IgG for passive transfer to rodents. In the frequently used accelerated version of NSN, the recipient is first immunized with IgG from the donor species to induce rapid development of an immune response to donor IgG. Because the model depends on deposition of, and in some cases immunity to, a planted foreign Ag, NSN does not provide insight into the origins of autoimmune GN. The Ag and epitopes recognized by anti-GBM IgG in nephrotoxic serum preparations are rarely well defined and may be distinct from those recognized by IgG of patients with anti-GBM GN and GP disease. Recent discoveries regarding the cryptic nature of GP Ag in rodents in particular suggest that this is often the case (38). Nonetheless, NSN models and informative mutants have provided invaluable insight into the numerous effector mechanisms that are engaged after antibody deposition on renal basement membranes and in generation of crescentic GN (reviewed in Refs. 23, 66). Studies comparing histopathologic features, leukocyte and renal cell subsets, cytokines, and various disease mediators in kidneys of rodents with NSN with those in kidneys of patients with crescentic GN suggest that similar mechanisms are involved in the different species.

ANCA-Associated Vasculitis

AAV is the most common type of crescentic GN and necrotizing vasculitis in adults. Its very high mortality if untreated and requirement for prolonged therapy with toxic agents due to a chronic relapsing and remitting course have infused urgency into the search for better treatments. Much research has focused on understanding the role of humoral immunity in AAV, in particular the significance and pathogenic role of circulating ANCA that are key to diagnosis. This focus may be justified by observations from a recent genome-wide association (GWAS) study that revealed that genetic associations in AAV track with ANCA serotype, not disease phenotype (39). These findings have bolstered support for the notion that anti-myeloperoxidase (MPO) and anti-proteinase 3 (PR3) AAV are distinct diseases with different etiopathogenesis. This notion also suggests that different animal models will be required to clarify mechanisms and that optimal interventions may not be interchangeable.

ANCA are detected in serum but generally not in tissues (hence the pauci-immune designation). Numerous in vitro studies have demonstrated the capacity of ANCA to interact with target Ag (MPO or PR3) expressed on the surface of primed human neutrophils and promote neutrophil activation and degranulation and endothelial cell injury. Confirmation of a pathogenic role in disease has required in vivo validation by animal modeling. The most significant advance to date was the development of an anti-MPO model that leverages MPO-deficient (MPO−/−) mice to produce high-titer high-affinity anti-MPO IgG (73). Whereas mouse MPO immunization alone does not induce AAV in mice, passive transfer of anti-MPO IgG raised in MPO−/− mice to wild-type or immunodeficient Rag−/− B6 recipients induces mild necrotizing crescentic GN (NCGN) at 1 wk. In a modified model, wild-type (MPO+) bone marrow is transferred into MPO−/− mice after recipient MPO immunization and irradiation; maintenance of high serum levels of anti-MPO IgG is attributed to radioresistance of anti-MPO plasma cells. The MPO chimeric mice develop prolonged NCGN that is variably more robust than after passive transfer (up to ∼30% glomeruli with crescents at 8 wk, compared with 10% 6 days after IgG transfer) (59, 60). Whereas adoptive transfer of anti-MPO splenocytes from immunized MPO−/− mice also induces AAV-like lesions, concurrent Ig deposition obfuscates attribution of pathogenesis in this variant (73).

The MPO−/− facilitated model was a major breakthrough in proving that anti-MPO IgG alone are sufficient to induce focal NCGN in naïve subjects. The model also proved sufficiently reproducible to serve as a platform to elucidate effector pathways, particularly in the context of early neutrophil-mediated injury of acute lesions. The model and iterations based on informative genetically engineered mutants revealed an essential role for neutrophils and bone marrow cell-derived MPO Ag (59, 74), disease amplifying effects of endotoxin (26), an unexpected requirement for the alternative complement pathway (75), and a C5a and myeloid cell C5aR amplification loop (60). Importantly, translatability has been supported by evidence of similar mediators in AAV patients.

Preclinical studies in the MPO−/− facilitated model have also demonstrated efficacy of several potential therapies, including multiple agents already in clinical use. Amelioration of NCGN was demonstrated for C5 mAb blockade, an oral phosphoinosatide 3-kinase-γ inhibitor, in vivo IgG deglycosylation, a proteasome inhibitor (bortezomib) that depletes anti-MPO plasma cells, and the IL-1 receptor antagonist anakinra (reviewed in Ref. 58). Capitalizing on the capacity of human C5aR (CD88) to respond to mouse C5a, Xiao et al. (72) recently demonstrated that an oral small molecule antagonist of human C5aR blocks induction of anti-MPO NCGN in 129S6/B6 mice bearing a transgenic human C5aR.

A notable limitation of the MPO−/− facilitated model is its failure to recapitulate the severe GN with renal failure observed in many AAV patients. Serum creatinine is not elevated in mice that receive anti-MPO IgG (73). Additionally, the model requires raising high-affinity MPO IgG in the absence of self-Ag. It thus has limited capacity to inform the field about the origin of spontaneous anti-MPO autoIg in patients, including the nature of genetic and environmental factors that promote loss of tolerance. Nonetheless, the ability of immunization to readily induce a high-affinity anti-MPO response only in MPO−/− mice supports the notion that MPO is the natural in vivo tolerogen.

Direct evidence for an in vivo pathogenic role for anti-PR3 has proven more difficult to demonstrate. Species differences in PR3 Ag structure, biology, and regulation form one barrier to in vivo modeling. In contrast to rodent MPO, which shares some target epitopes with human MPO, mouse PR3 does not express human ANCA pathogenic epitopes (27). Neither immunization with human nor mouse PR3 induces anti-mouse-PR3 antibodies (53), and wild-type mice injected with patients' anti-PR3 ANCA fail to develop disease (35). Moreover, mouse PR3 is not expressed on the surface of mouse neutrophils, in contrast to the situation in humans. Plasma membrane localization of secreted PR3 (or MPO) is thought to be a critical event in promoting ANCA binding and activation of primed neutrophils. Lipid membrane anchoring by human PR3 is attributed to a hydrophobic amino acid patch, as well as binding to the GPI-anchored membrane protein NB1/CD177 (31). Mouse PR3 is hydrophilic in this region, suggesting it lacks a critical membrane insertion domain. Thus, even if anti-mouse-PR3 autoreactive IgG are induced in vivo, absence of neutrophils bearing membrane PR3 would prevent development of AAV. This presumably explains why passive transfer of anti-mouse-PR3 antiserum raised in PR3−/− mice fails to induce disease in recipients, unlike the case using MPO−/− mice, despite prolonged maintenance of high serum levels of anti-PR3 IgG (53). A novel human PR3 transgenic mouse that demonstrates expression of human PR3 on activated mouse neutrophils has been generated, although only preliminary characterization is reported (24); this model may circumvent the problems with mouse PR3, assuming that the transgenic human Ag can be expressed on the mouse neutrophil surface.

The species-specific roadblocks led Little et al. (35) to explore a humanized model of anti-PR3 disease. Transfer of patients' PR3-ANCA IgG into endotoxin-primed Hu-HSC mice, created using healthy donor (PR3+) HSC, induced mild disease. Hematuria, glomerular hypercellullarity, and pulmonary capillaritis were observed in a subset of mice at day 6. Although limited in scope, this is the first experimental evidence of an in vivo pathogenic role for human anti-PR3 IgG. Notably, design and technical limitations, including dependence on a single HSC donor and low level human chimerism, including low level neutrophil reconstitution and near absence of human T cells, may have prevented full disease expression. Repeat attempts are warranted, noting that a successful model may require maneuvers that enhance human neutrophil PR3 expression or apoptosis.

PR3-ANCA and MPO-ANCA AAV appear to differ fundamentally in multiple aspects. A GWAS revealed an association between a PR3 gene polymorphism and PR3-ANCA that was not seen between MPO and MPO-ANCA (39). PR3 expression is dysregulated in neutrophils of patients (9). Millet et al. (45) suggest that dysregulated PR3 expression in AAV extends to apoptotic neutrophils, which express elevated levels of phosphatidylserine-associated PR3 on their membrane. These investigators propose a novel paradigm in which macrophage ingestion of PR3+ apoptotic neutrophils shifts an anti-inflammatory microenvironment to a pro-inflammatory one dependent on nitric oxide and IL-1β production. This PR3-dependent response supports Th2/Th9 and Th1 CD4 T-cell activation in the absence of PR3-ANCA but is skewed toward a Th17 response in the presence of PR3-ANCA. These novel observations will need to be taken into consideration during development of PR3-ANCA animal models.

Summary

Animal models of human GN remain indispensible in shaping our understanding of pathogenesis of GN and in evaluating novel therapies. The complexity of in vivo microenvironments that generate and modulate immune responses cannot be adequately modeled in vitro or in silico. Technological advances are permitting development of new models that not only closely replicate morphologic and clinical hallmarks of human disease but that also express human Ag and susceptibility genes and that recapitulate human-specific ligand-receptor interactions. Selective humanization can circumvent species-specific barriers while preserving cross-species compatibilities. Humanized models have permitted fresh insight into pathogenesis of anti-GBM GN and IgAN and provided a platform for preclinical efficacy testing of a potential new treatment for MPO-ANCA vasculitis and will hopefully be available soon to accelerate discovery in PR3-ANCA vasculitis and primary MN. Hu-HSC models will need continued refinement and careful interpretation, with consideration of cross-species differences. Additional tools, such as affordable recombinant species-specific Ag and soluble probes to select Ag-specific lymphocytes, will facilitate model development. Multiple models of each disease will likely be needed. The expectation that a single model, strain, or monoclonal antibody must recapitulate the entire spectrum of a human disease has been rendered obsolete. Rather, model systems that take into account the genetic heterogeneity of outbred human populations are needed. Individual models are likely to replicate only a limited aspect of disease or a subset of patients, with selected genetic and exposure backgrounds and other factors that ultimately determine immune and inflammatory responses. There is also an urgent need for models to explore the origins of spontaneous anti-kidney autoimmunity, relapsing disease, and the role of environmental factors in the onset and propagation of GN (44, 77). As improved models are developed, investigative focus will necessarily and appropriately continue to shift between animals and patients, as novel findings in one arena inform the other and they collectively lead to new therapies.

GRANTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-088904 and P30-DK-096493, National Institute of Environmental Health Sciences Grant R21-ES-024451, Institute for Medical Research, and Durham Veterans Affairs Medical and Research Services.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).